Biomedical Engineering Reference
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An example of an ultrasound image of an adult rhesus testis is shown in Fig. 11.3b ,
where the rete testis space is visualized as an echodense line. An injection needle
(22 Ga) is inserted into the testis trans-scrotally and the tip of the needle is placed
within the rete testis space (Fig. 11.3b ). Ultrasound-guided rete testis injection is
percutaneous, and, thus, is considered a noninvasive procedure because the injection
needle is simply passed through the scrotum into the testis. To illustrate this tech-
nique, the vital dye trypan blue was injected into rhesus macaque testes using this
approach and testes were recovered immediately after injection by castration. After
recovery, trypan blue dye could be visualized in the ductules of the caput epididymus,
which is contiguous via the efferent ducts with the rete testis, confirming the success
of these injections (Fig. 11.3c ). Bisection of the transplanted testis revealed that blue
dye radiated from the rete testis into approximately 60-80% of seminiferous tubules
(Fig. 11.3d ). Subsequent evaluation of intact seminiferous tubules confirmed the pres-
ence of blue dye in the lumen of seminiferous tubules (Fig. 11.3e, f ). Thus, ultra-
sound-guided rete testis injection can be used to introduce donor SSCs into infertile
recipient testes to test the regenerative potential of primate SSCs.
Dobrinski and co-workers have demonstrated the feasibility of performing SSC
transplants in various other large animal models using ultrasound-guided rete testis
injection (Honaramooz et al. 2002, 2003a, b ; Kim et al. 2008 ). In goats, trans-
planted donor cells ultimately produced complete spermatogenesis, established
fertility and transmitted the donor haplotype to progeny (Honaramooz et al. 2003b ).
Furthermore, transplants by ultrasound-guided rete testis injection were success-
fully performed between out-bred, immune competent goats and dogs, suggesting
that the testis is an immune privileged site (D'Alessio et al. 2001, 2004 ; Kim et al.
2008 ).
In primates, injection of dye into seminiferous tubules using ultrasound-guided
rete testis injection was initially performed in cynomolgus monkey and human
testes, ex vivo (Schlatt et al. 1999 ). Furthermore, when donor germ cells preloaded
with BrdU were transplanted into recipient monkey testes, labeled B-spermatogonia
could be identified in recipient seminiferous tubules four weeks after transplant,
demonstrating successful engraftment of donor cells into recipient monkey testes
(Schlatt et al. 1999 ). Similar injections to assess SSC engraftment and spermato-
genic regeneration have also been described in adult cynomolgous monkeys
( Macaca fascicularis ) that received 2 Gy of X-radiation (Schlatt et al. 2002a ).
In this study, transplanted testes exhibited modest increase in testis volume
compared to contralateral testis (sham transplanted) during the 9 months after trans-
plant (Schlatt et al. 2002a ). Remarkably, the testes of two animals exhibited com-
plete spermatogenic recovery. Despite promising results in these autologous SSC
transplants, it was impossible to definitively identify donor spermatogenesis
because donor cells were not marked. Thus, spermatogenesis arising from trans-
planted SSCs and recovering endogenous spermatogenesis could not be discrimi-
nated because the cells were identical. Similar autologous SSC transplants were
performed in juvenile rhesus macaques that received 10 Gy radiation (Schlatt et al.
2009 ), but again, results of these transplant were equivocal since donor cell engraft-
ment could not be definitively identified.
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